KR102027790B1 - Method for isolation and purification of human oligosaccharide using lactose removal of immobilized Kluyveromyces lactis - Google Patents

Abstract

The present invention relates to a method for isolating and purifying breast milk oligosaccharides using lactose removal ability of immobilized Cluyveromyces lactis, which simplifies the process by using immobilized Cluyveromyces lactis and distilled water to shorten the time. In addition, by selectively removing only lactose from breast milk, it is expected that the breast milk oligosaccharides can be efficiently separated and purified without changing the concentration of the breast milk oligosaccharides, and thus can be usefully used in the breast milk oligosaccharide related industry.

Description

Method for isolation and purification of human oligosaccharide using lactose removal of immobilized Kluyveromyces lactis}

The present invention relates to a method for isolating and purifying lactose-free breast milk oligosaccharides without changing lactose oligosaccharides utilizing the excellent lactose removal ability of immobilized Kluyveromyces lactis.

Human milk oligosaccharides (HMOs) comprise 5-15 g per liter of human milk. This is similar to the amount of protein in breast milk and in excess of the amount of lipids. HMO is five, including D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc) and sialic acid (Sia; N-acetyl neuraminic acid [Neu5Ac]) Monosaccharides are present and generally share these major elements and have terminally lactose (Gal1-4Glc) residues. Galactose of lactose residues forms 3'- sialic lactose or 6'- sialic lactose through α- (2, 3) and α- (2, 6) bonds, and α- (1, 2) and α- It is fucosylated with (1, 3) linkages to form 2'-fucosyllactose and 3'-fucosyllactose. The structure of the HMO having such a basic form repeatedly includes N-acetyllactosamine (Gal1-3 / 4GlcNAc) units in order to have complexity, and consists of different components. This results in more than 130 HMOs in breast milk.

Physiological characteristics of HMO include prebiotic effect, intestinal adhesion inhibition of pathogens, and immune control system regulation. Prebiotic effect has been studied the most so far. HMO in breast milk is growing bifidobacteria in the dominant species in the early chapters of infant gambling Te Solarium BP bonus (Bifidobacterium bifidum ) selectively promotes growth, but cannot be used by harmful bacteria. In addition, HMOs have been known to inhibit intestinal adhesion of pathogens, because the cell wall polysaccharide structure of pathogens that bind intestinal lectins often resembles some structures of HMO, resulting in HMOs providing water-soluble ligand analogs. Breastfeeding infants are resistant to infection by pathogens. In addition, HMO is partially absorbed in the intestine of the infant and excreted in the urine of breastfeeding infants, where some HMO enters the body circulation and is involved in the protein-carbohydrate interaction involved in the white blood cell rolling process and platelet formation in the vessel wall. It is expected to change relationships and consequently regulate the immune system. However, although HMOs and infants' immunity are expected to be deeply correlated with each other, full-scale experimental results are not yet sufficient, because it was difficult to provide enough single HMOs for the experiment. In addition, the complexity and diversity of HMO structures make chemical synthesis difficult, and thus, transformation methods have been developed that can replace oligosaccharides obtained from other species or express human glycosyltransferase in animals. However, oligosaccharides of other species differ in composition or structure from HMOs, and oligosaccharides produced from transgenic animals need to be evaluated for their effects on human physiological activity. Due to this HMO specificity and difficulty in synthesis, techniques for mass separation and production of HMO from breast milk are essential for various analyses.

Breast milk makes up 30-40% of lactose, making HMO purification difficult. Existing HMO purification methods include size-exclusion chromatography, simulated moving bed chromatography, and coupling flash liquid chromatography. However, chromatographic purification requires expensive costs and requires expert skill in the method. Therefore, in order to solve the above problems, a new HMO purification technology is required for cost reduction and convenience of operation for HMO purification.

Kluyveromyces lactis , K. lactis ) is a migratory yeast with a haploid life history and many subspecies belonging to this species have been found in dairy products. Β-galactosidase of K. lactis can be broken down into glucose and galactose by breaking the β- (1,4) and β- (1,3) bonds of lactose. However, in order to use β-galactosidase in the purification of oligosaccharides, enzymes must be separated by electrophoresis or the like. After the β-galactosidase treatment, the fermentable sugar is removed by another yeast or filtration method. It has the complexity of doing it. K. lactis is able to consume lactose and monosaccharides through glycolysis. Therefore, the present invention is to determine the lactose consumption of K. lactis in order to obtain high purity HMO in breast milk, and to determine the effect of HMO purification after fixation of K. lactis using calcium alginate.

On the other hand, Korean Patent No. 1122928 discloses a method for producing lactose-depleted milk to remove lactose by coagulating degreasing milk solution, but breast milk oligosaccharides using lactose removal ability of the immobilized Kluyveromyces lactis of the present invention. There is no disclosure of separation and purification methods.

The present invention is derived from the above-mentioned demands, and identifies an optimal method for selectively removing lactose and lactose degradation products from breast milk using immobilized Kluyveromyces lactis, and the mother milk separated and purified by the above method. The present invention was completed by confirming that lactose was removed from oligosaccharides without changing the concentration of breast milk oligosaccharides.

In order to solve the above problems, the present invention

1) immobilizing Kluyveromyces lactis with alginate;

2) activating the immobilized Kluyveromyces lactis by immersing the immobilized Kluyveromyces lactis of step 1) in the culture medium;

3) removing the lactose contained in the mother milk by adding the activated immobilized Kluyveromyces lactis of step 2) to the mother milk; And

4) separating and purifying the mother milk oligosaccharide from the lactose-free lactose of step 3) provides a method for isolating and purifying lactose-free breast milk oligosaccharides without changing the breast milk oligosaccharides.

The present invention also provides a milk milk oligosaccharide from which the lactose separated and purified by the above method is removed.

The present invention can selectively remove lactose from breast milk by using K. lactis , and the total fermentable sugars (lactose, glucose and immobilized by high concentration of immobilized Kluyberomyces lactis ). Galactose) shortens the fermentation time required for removal, and the recovery of the immobilized cells can be simplified to simplify the process, and the overall process time can be shortened by omitting the preculture time of the cells. In addition, by eliminating lactose by directly fermenting the high concentration of immobilized Cluyveromyces lactis in mother's milk in distilled water, there is an effect that can easily proceed to the purification process of breast milk oligosaccharides without a separate process for removing the medium.

1 is a result of culturing by adding glucose or lactose to YP medium and confirming the concentration of growth (a), substrate (b) and product (c) of K. lactis .
Figure 2 is a result of confirming the concentration of the growth (a, b), substrate (c, d) and product (e, f) of K. lactis according to the medium and carbon source. As the medium, YP medium (a, c, e) and YNB medium (b, d, f) were used, and the carbon source was glucose, lactose and galactose, respectively.
Figure 3 is the result of confirming the concentration of the growth (a), substrate (b) and product (c) of K. lactis yeast according to the amino acid content. W / O is YNB medium without amino acid, 1% is YNB medium without amino acid, and 1% (v / v) is mixed with YNB medium of the same concentration containing amino acid, and 5% is amino acid 5% (v / v) of the same concentration of YNB medium containing amino acids in the YNB medium containing no amino acid, and 10% of the same concentration of YNB medium containing amino acids in the YNB medium containing no amino acids 10% (v / v) is a mixed medium, 100% is the same concentration of YNB medium containing amino acids.
Figure 4 is a result of checking the concentration of the growth of K. lactis in accordance with the content and the amino acid concentration of the K. lactis (a), the substrate (b) and the product (c). W / O is YNB medium without amino acids, 5% is YNB medium without amino acids, and 5% (v / v) of YNB medium with the same concentration of amino acids is contained in 10% amino acids. This is a medium in which 10% (v / v) of the same concentration of YNB medium containing amino acid is contained in the YNB medium not containing this.
5 is a result of confirming the concentration of growth (a), substrate (b) and product (c) of K. lactis with or without the presence of the medium. without broth is a distilled water treatment group, YNB W / O_a.a is a YNB medium treatment group does not contain amino acids.
6 is a result of confirming the concentration of the substrate and product by the activation of the immobilized K. lactis . (a) is the activated immobilized K. lactis treated group, and (b) is the inactivated immobilized K. lactis treated group.
7 is a result of confirming the concentration of growth (a), substrate (b) and product (c) of the immobilized K. lactis according to the amino acid content. W / O is YNB medium without amino acid, 1% is YNB medium without amino acid, and 1% (v / v) is mixed with YNB medium of the same concentration containing amino acid, and 5% is amino acid 5% (v / v) of the same concentration of YNB medium containing amino acids in the YNB medium containing no amino acid, and 10% of the same concentration of YNB medium containing amino acids in the YNB medium containing no amino acids 10% (v / v) is a mixed medium, 100% is the same concentration of YNB medium containing amino acids.
8 is a result of confirming the concentration of the substrate (a) and product (b) of the immobilized K. lactis with or without the presence of the medium. without YNB is a group treated only with distilled water without a medium.
Figure 9 is the result of confirming the fermentation sugar removal ability of immobilized K. lactis yeast in lactose, GAC (galactooligosaccharides) or primary purified breast milk oligosaccharides. (a) is the concentration of lactose and (b) is the concentration of the product.
10 is a result confirming the loss of GOS and breast milk oligosaccharides after fermentation by immobilized K. lactis . (a) is a graph of the area of DP (degree of polymerization) 2 shown in the ion chromatography results, and (b) is a result of confirming the relative strength of oligosaccharides of DP3 or more according to the fermentation time.

The present invention

1) immobilizing Kluyveromyces lactis with alginate;

2) activating the immobilized Kluyveromyces lactis by immersing the immobilized Kluyveromyces lactis of step 1) in the culture medium;

3) removing the lactose contained in the mother milk by adding the activated immobilized Kluyveromyces lactis of step 2) to the mother milk; And

And 4) separating and purifying the breast milk oligosaccharides from the lactose-removed breast milk of step 3). The present invention relates to a method for separating and purifying lactose-removed breast milk oligosaccharides without changing the breast milk oligosaccharide.

In one embodiment of the present invention, the immobilization of Cluiveromyces lactis of step 1) is mixed with 0.5 ~ 1.5 parts by weight of sodium alginate per 1 part by weight of Cluiveromyces lactis from which the culture medium is removed and dropped into calcium chloride solution It is to be immobilized by stirring, more preferably 1 part by weight of sodium alginate per 1 part by weight of Cluyveromyces lactis, and then immobilized by dropping it in a calcium chloride solution, but is not limited thereto.

The immobilized Kluyveromyces lactis can be stored in an inactive state by immersing in low-temperature calcium chloride solution during long-term storage, and the immobilized Kluyveromyces lactis of the inactive state can be activated by immersing in culture medium for 11-13 hours. More preferably, it may be used for fermentation after activating by immersing in culture medium for 12 hours.

In one embodiment of the present invention, the mother milk of step 3) may be used unrefined mother milk and refined mother milk, but preferably using a primary purified mother milk.

Breast milk can be purified using the Folch partition method and cold ethanol precipitation,

More preferably

1) centrifuging breast milk at 4500C for 30 minutes at 4 ° C;

2) collecting only the intermediate layer of the sample centrifuged in step 1);

3) 4 parts by weight of a mixed solution of chloroform and methanol in a volume ratio of 2: 1 per 1 part by weight of the intermediate layer collected in step 2) were added, and centrifuged for 30 minutes at 4500 rpm at 4 ° C. Doing;

4) collecting only the supernatant from the centrifuged sample of step 3), add 3 parts by weight of ethanol per 1 part by weight of the supernatant, and react at 4 ° C. for 24 hours;

5) collecting the supernatant by centrifuging the reaction product of step 4) at 4 ° C. at 4500 rpm for 30 minutes; And

6) lyophilizing the supernatant of step 5); but may be purified, including, but not limited to.

In one embodiment of the present invention, the separation and purification of the breast milk oligosaccharide of step 4) is through centrifugation, filtration and lyophilization, but is not limited thereto.

'Removal of lactose' of the present invention preferably means the removal of lactose and lactose degradation products, the lactose degradation products being, but not limited to, glucose, galactose and ethanol.

In a preferred embodiment of the present invention, the method for separating and purifying breast milk oligosaccharides to remove lactose from the mother milk is

1) Kluyveromyces with culture removed lactis ) mixing 1 part by weight of alginate per 1 part by weight, and then dropping the result in a calcium chloride solution to fix it;

2) activating the immobilized Kluyveromyces lactis by immersing the immobilized Kluyveromyces lactis of step 1) in the culture medium for 12 hours;

3) removing the lactose contained in the mother milk by adding the activated immobilized Kluyveromyces lactis of step 2) to the primary purified mother milk dissolved in water; And

4) separating and purifying the mother's milk oligosaccharide by centrifugation, filtration and lyophilization in the lactose-removed mother milk of step 3), but may not be limited thereto.

In addition, the present invention provides a mother milk oligosaccharide from which lactose has been separated and purified by a method of separating and purifying mother's milk oligosaccharides from which lactose is removed from the mother milk.

The breast milk oligosaccharides are selectively removed from lactose and lactose decomposition products from breast milk, and there is no change in the concentration of breast milk oligosaccharides, but is not limited thereto.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Experiment method

1. Use strains and culture conditions

K. lactis was used as a strain stored in the fermented food science laboratory at Chungnam National University. K. lactis was prepared using prepared YPD medium (yeast extract [Bacto, New Jersey, USA] 10 g / l, Peptone [Bacto, New Jersey, USA] 20 g / l, Dextrose [Sigma-Aldrich, USA] 10 g / l). Incubated at a temperature of 30 ℃, was used in the experiment while storing at -80 ℃ in 15% (v / v) glycerol stock (glycerol stock). The strain was inoculated in YPD medium using an Optizen 2120 UV spectrophotometer (Mecasys, Seoul, Korea) to have an optical density (OD) of 0.1. The culture was incubated in aerobic conditions in a rotary shaker (Hanbaek, Gwangiu, Korea) for smooth oxygen inflow.

2. According to the culture conditions K. lactis Growth measurement of free yeast

The growth curve of K. lactis, the removal of sugars and the production of metabolites were examined for the effects of external environmental conditions.

In order to confirm that K. lactis can remove lactose (lactose), 10 g / L of glucose and lactose were added to YP medium, and then K. lactis was inoculated and cultured.

The effect on the type of medium and sugar was inoculated and cultured with K. lactis after adding 20 g / l of glucose, lactose and galactose to YP medium and YNB medium, respectively.

The effect of amino acids is to mix YNB medium and YNB W / O a / a (without amino acid) medium in W / O (without), 1%, 5%, 10%, 100% (v / v) depending on the amino acid ratio. After inoculation, 60 g / l lactose was added to inoculate K. lactis and cultured.

Effect of initial inoculation density of K. lactis on a 1.5% by K. lactis in a culture medium containing lactose and 20g / ℓ for the average concentration of inoculation (v / v) was inoculated with 25% of the culture medium for inoculation at a high concentration (v / v K. lactis Only pellets were inoculated and incubated.

The effect of the medium was incubated in YNB W / O a / a medium or distilled water to which 40 g / L of lactose was added and then cultured. The effect of sugar isolated from breast milk was inoculated and cultured with K. lactis in YNB W / O a / a medium containing 40 g / L of lactose and breast milk sugar isolated.

The volume of the culture solution was all 200 ml, shaking culture was carried out at 30 ℃ 200 rpm. Cell growth was confirmed by measuring absorbance at 600 nm every three hours. Sugar isolated from breast milk (primary refined breast milk) was obtained by treating the milk donated by Maeil Dairy by the Folch partition method and cold ethanol precipitation.

3. Calcium Alginate (calcium alginate)  Used K. lactis fixing

K. lactis to be used for immobilization was inoculated in 200 ml of YPD medium and incubated for 12 hours at 30 ° C. and 200 rpm. Subsequently, the culture solution was centrifuged at 4 ° C. and 4500 rpm for 10 minutes using a medical refrigeration centrifuge (vision scientific, Gyeonggi, Korea) to remove the culture solution, and the same amount of the 1% (v / v) yeast extract mixture (enzyme digestion). By mixing the yeast extract concentrated in the water-soluble portion of the enzyme into distilled water) to remove the secondary centrifugation and the medium. The medium removal and washing process was repeated three times. The yeast solution from which the culture solution was removed was uniformly mixed at the same ratio as 3% (w / v) sodium alginate, and then dropped into 1 L of 0.5M CaCl ₂ using a syringe (syringe, Koreavaccine, Gyeonggi, Korea). I dropped it in a state. Alginate beads were stabilized in 0.5M CaCl ₂ solution for at least 2 hours for fermentation and used for fermentation, and stored in 4 ° C. CaCl ₂ solution for long time storage. The immobilization process was repeated two times to obtain a K. lactis immobilized product corresponding to a total of 400 ml of YPD medium.

4. According to the culture conditions K. lactis Growth Measurement of Immobilized Yeast

K. lactis For inactivation of immobilized yeast, inoculated in YNB medium containing 20 g / l of lactose, followed by shaking culture at 100 ° C. at 30 ° C. for 12-14 hours. K. lactis Immobilized yeast was washed with distilled water for use in experiments after activation.

The effect of K. lactis immobilized yeast activation was confirmed by inoculation and incubation of inactivated immobilized yeast stored in CaCl 2 and activated immobilized yeast in YNB medium in 200 ml of YNB medium to which lactose 20g / L was added.

The effect of medium amino acid concentration on lactose 20g / L and YNB medium and YNB W / O a / a (without amino acid) medium were W / O (without), 1%, 5%, 10%, 100% according to amino acid ratio. It was confirmed by inoculation and incubation in 100 ml of YNB medium mixed with (v / v).

To determine whether the medium was affected by high concentrations of sugar, K. lactis was added to YNB medium with lactose 40 g / L or distilled water with 40 g / L lactose. Immobilized yeast was inoculated.

K. lactis In order to confirm the effect on the complex sugar of immobilized yeast, inoculated and incubated in distilled water containing 20 g / L of GOS, primary purified milk or lactose, respectively.

5. K. lactis of  Determination of sugar consumption and metabolite production concentration

After centrifugation, the supernatant was detected by metabolites using a high-pressure liquid chromatography infinity 1260 (Agilent Technologies, USA) with a refractive index (Agilent Technologies, California, USA). The column used Rezex Roa-organic acid H + mm (Phenomenex, California, USA), the mobile phase was flowed at a flow rate of 0.6ml / min with 0.005N sulfuric acid, and analyzed under a temperature condition of 40 ℃. Samples were diluted to 1/10 and 40 μl sample was injected once. Standards for measurement were glucose, galactose, lactose, lactate and ethanol.

6. HMO  Measuring loss

Breast milk oligosaccharides and GOS cultures were measured by washing with Porous graphitic carbon-Solid phase extraction (PGC SPE). Samples obtained at 0, 4, 8 and 12 hours were separated using UPLC (Ultra Performance Liquid Chromatography) and measurements were detected using the scan mode of the 6550 Quadrupole time-of-flight mass spectrometer (Q-TOF). The column was porous graphitic carbon, and 3% ACN (acetonitrile) with 0.1 FA (ammonium formate) and 90% ACN with 0.1 FA were used as the mobile phase.

Example  1. Environmental conditions for culture K. lactis on  Effect on the removal of fermentable sugars

1) Removal of lactose and glucose by K. lactis

K. lactis was cultured in medium adjusted by adding 10 g / l glucose or lactose to YP medium, and cell growth and substrate and product concentrations were measured.

As a result, as shown in Figure 1 (a) K. lactis in YP medium showed the maximum cell growth after 18 hours of fermentation, OD value of 8.2 when added glucose, 7.5 OD added by lactose added glucose to 7.5 In this case, it was about 10% higher than the addition of lactose.

In addition, after 15 hours of fermentation, the concentrations of glucose and lactose converged to 0, but the concentrations of the products were different. The main products through the culture of lactose and glucose of K. lactis were ethanol, and the concentrations of ethanol and lactate increased to 11.25 g / l and 1.13 g / l, respectively, after glucose culture (Fig. 1 (b)). In use, ethanol and lactic acid concentrations were 14.96 g / l and 1.40 g / l, respectively (FIG. 1 (c)).

Through the above results, it was confirmed that after fermentation, both glucose and lactose were removed.

2) Metabolic changes of K. lactis according to medium and carbon source

Given the downstream process of purifying oligosaccharides after fermentation, it is advantageous to minimize and simplify the components of the medium. Therefore, YP medium, which is a rich media, was replaced with YNB, which is a defined media, to compare the fermentable sugar removal ability of K. lactis .

Glucose, lactose and galactose were added to the YP medium and the YNB medium at 20 g / L, respectively, followed by incubation of K. lactis . As a result, as shown in Figure 2 the components of the medium and the type of sugar K. lactis It affected growth. Maximum cell concentrations in YP medium were OD 7.84, 6.98 and 5.78, respectively, in the order of glucose, lactose and galactose addition. The addition of lactose and galactose showed 90% and 75% of the maximum cell growth concentration using glucose, respectively (FIG. 2 (a)). When YNB medium was used, the maximum cell growth concentration of K. lactis was decreased compared to YP medium. The maximum absorbance of YNB medium with glucose, lactose and galactose was OD 5.41, 5.30 and 3.50, which was 60-70% compared to when fermented in YB medium using the same carbon source (Fig. 2 (b)). ).

In addition, the substrate removal and product production also showed a difference depending on the medium and sugar. Glucose, lactose and galactose at 20 g / L in YP medium, while glucose and lactose reached 0 g / L after 24 hours, galactose disappeared after 28 hours (Fig. 2 (c)). The ethanol concentrations produced were all 11.32 g / l (FIG. 2 (e)). After 24 hours of incubation in YNB medium, the concentration of glucose and lactose converged to 0, but the concentration of galactose was 8.57 g / L (FIG. 2 (d)). Ethanol production of glucose, lactose and galactose was also the lowest value in galactose at 9.60 g / l, 10.65 g / l and 5.26 g / l, respectively (FIG. 2 (f)). The consumption rate of galactose was about 60% compared to glucose and lactose, but when the fermentation time was extended for 36 hours, all the galactose in the medium disappeared.

3) Changes in Efficacy of K. lactis According to Amino Acid Content

The YNB medium (YNB w / o Amino acid) containing no amino acid and the conventional YNB medium were used to confirm the correlation between the lactose removal of K. lactis and the amino acid concentration in the medium. After mixing the same concentration of the existing YNB medium in the YNB medium containing no amino acid at 0%, 1%, 5%, 10%, 100% (v / v), after adjusting the lactose concentration to 60g / l, K. culturing the growth of K. lactis lactis and subsequent reduction of the lactose due, it confirmed the generation result of the fermentation by-products.

As a result, as shown in FIG. 3, the maximum absorbance of the total medium in which the amino acids were adjusted differently was 4.99 (± 0.09), and there was no significant difference in each medium (FIG. 3 (a)). Lactose removal and ethanol production did not show any amino acid differences. The concentration of 24 hours after lactose and ethanol are each 35.51 (± 1.18) g / ℓ , 14.72 (± 1.01) a g / ℓ shown to be similar in all the amino acid medium in the medium concentration effect on the removal of sugar fermentable K. lactis It was confirmed that did not have (Fig. 3 (b, c)).

4) Elimination of Fermentative Sugars Using High-Concentration K. lactis Culture

To determine the effect of initial K. lactis concentration on the removal of fermentable sugars, lactose was added to YNB medium having different amino acid concentrations, and then cultured by inoculating K. lactis at high and normal concentrations. The maximum cell concentration after fermentation for 15 hours at high concentration was OD 4.76 (± 0.33), and the maximum cell concentration after fermentation at normal concentration reached OD 4.04 (± 0.11) after 21 hours (Fig. 4 (a)).

In addition, it took 18 hours for high concentration inoculation and 22 hours for general inoculation for K. lactis to remove 20g / l lactose. At the end, the concentration of lactose was 0.80 (± 0.42) g / l and most of the lactose was removed, and the concentration of ethanol produced was all around 10 g / l (Fig. 4 (b, c)). Except for fermentation time, there was no difference in the removal of fermentable sugars and the concentration of final metabolite.

5) Effect of Media on Fermentative Sugar Removal

If K. lactis is used to remove fermentable sugars, the fermentation sugars should be consumed without adding other ingredients to the medium, considering the oligosaccharide purification after the fermentation process. Therefore, only the fermentable sugars were added without adding the medium to confirm the extent to which K. lactis consumed the fermentable sugars.

40g / L of lactose was added to YNB medium without amino acid and distilled water, respectively, and then cultured by inoculation with activated K. lactis in YNB medium.

As a result, as shown in Figure 5 (a) K. lactis growth showed a significant difference depending on the addition of the medium. The maximum cell concentration was OD 5.64 when incubated in YNB medium without amino acids. Somatic cell division was hardly observed when K. lactis was inoculated in distilled water, so the maximum absorbance was OD 0.46. However, lactose removal and ethanol production were not affected by the medium (Fig. 5 (b, c)). The final concentrations of lactose and ethanol were 13.66 g / l and 11.29 g / l, respectively, after incubation with YNB medium, and the final concentrations were 13.26 g / l and 11.43 g / l, respectively.

Example  2.Fixing K. lactis  Of fermentable sugars using

1) immobilization Effect of K. lactis on Fermentation of Sugars

To develop an optimal process for the removal of fermentable sugars, K. lactis was immobilized at high concentration using alginate. Fermentation using a high concentration of immobilized yeast can shorten the fermentation time required to remove the total fermentable sugar, simplify the process by easily recovering the immobilized cells, and can shorten the overall process time by omitting the entire incubation time of the cells. There is an advantage.

Immobilized K. lactis stored in calcium chloride solution for a long period of time loses its metabolic activity and can lose or reduce fermentable sugar removal ability. To confirm this, immobilized K. lactis was stored at 4 ° C. for 3 days and then used in the experiment. Immobilization of K. lactis Immobilized K. lactis was used for 12 hours immersion in YNB medium before use in experiments. K. lactis activated before incubation Immobilized yeast and inactivated immobilized yeast were added to YNB medium containing 20 g / l of lactose, and the change of lactose concentration was observed.

As a result, as shown in FIG. 6, activation of immobilized K. lactis significantly affected lactose removal and ethanol production rate. When activated immobilized K. lactis was used, 20 g / l of lactose disappeared within 6 hours, and 8.96 g / l of ethanol was produced as a fermentation by-product (FIG. 6 (a)). However, in the case of using the immobilized K. lactis which was not activated for a long time, 6.65 g / l of lactose remained after 12 hours, and the concentration of ethanol was 2.18 g / l (Fig. 6 (b)).

Based on the above results, it was confirmed that the activation of immobilized K. lactis before fermentation was an important factor for the removal of fermentable sugars.

2) Effect of Amino Acids on the Removal of Fermentative Sugars of Immobilized K. lactis

The effect of amino acids on the removal of fermentable sugars of immobilized K. lactis was determined using YNB without amino acid and normal YNB medium. 1%, 5%, 10%, 100% (v / v) of normal YNB medium is mixed in YNB medium without amino acid (YNB without amino acid), and 20 g / l lactose is added and immobilized K. lactis Was inoculated and incubated to determine growth, lactose and ethanol concentrations.

As a result, as shown in Fig. 7 (a), the growth of immobilized K. lactis was hardly affected by the amount of medium amino acids. However, after 8 hours of fermentation, the concentration of lactose was decreased in proportion to the concentration of general YNB medium. In case of 100% of general YNB medium, 20g / L of lactose was consumed after 8 hours of reaction, but 4g / L of lactose remained in YNB medium without 1% or amino acid. However, after 10 hours, 20 g / l of lactose was consumed regardless of the content of general YNB medium. The concentration of ethanol produced was also constant at 8.13 (± 0.92) g / l (Fig. 7 (b, c)).

3) Effect of Medium on the Removal of Fermentative Sugars of Immobilized K. lactis

After adding 40 g / L lactose to distilled water containing no general YNB medium or medium components, activated immobilized K. lactis was added to confirm lactose removal and ethanol production.

As a result, as shown in Figure 8 lactose concentration after incubation for 12 hours was 0.27 (± 0.22) g / ℓ irrespective of the presence of YNB medium, most of the lactose was consumed (Fig. 8 (a)), ethanol In production, the concentration after culture was the same as 15.03 (± 0.10) g / l (Fig.

To the result of the experiment, using an immobilized K. lactis remove fermentable sugar mixed in the breast milk oligosaccharides was confirmed that this reaction can proceed directly from distilled water to activate the immobilized K. lactis.

4) Removal of Fermentative Sugars from GOS or Breast Milk Oligosaccharides Using Immobilized K. lactis

20 g / l of each of the primary purified mother's milk oligosaccharides containing lactose, GOS and lactose were dissolved in distilled water, and then fermented sugar was removed using activated immobilized K. lactis .

As a result, as shown in Figure 9, the initial 20 g / L lactose was all disappeared after 12 hours, 15 g / L lactose present in the primary purified breast milk oligosaccharides were all consumed after 8 hours. 4 g / L of galactose present in GOS disappeared after 6 hours. The ethanol concentrations after the reaction were 7.94 g / l, 2.81 g / l, and 6.75 g / l in lactose, GOS, and primary purified milk oligosaccharides, respectively.

In addition, as a result of confirming the loss of GOS and breast milk oligosaccharide after the reaction, as shown in Figure 10, the various oligomers (GOS) of the oligomer is a oligomer of DP (degree of polymerizatio) 2 with galactose by K. lactis during the fermentation process. Galactobiose was consumed, but no decrease in oligosaccharides above DP3 was observed. HMO was not found to change the concentration of oligosaccharides other than lactose. In particular, the concentration of fucosyllactose (DP3), the smallest oligosaccharide except lactose, did not change, and it was confirmed that fermentable sugars were selectively removed from breast milk oligosaccharides using immobilized K. lactis .

Claims (10)

1) Kluyveromyces lactis with culture removed Mixing 0.5 to 1.5 parts by weight of sodium alginate per 1 part by weight and dropping it in a calcium chloride solution to fix Cluyveromyces lactis;
2) activating the immobilized Kluyveromyces lactis by immersing the immobilized Kluyveromyces lactis of step 1) in the culture medium for 11 to 13 hours;
3) The activated immobilized Kluyveromyces lactis of step 2) is poured into a lipid extract of chloroform-methanol (2: 1, v / v) and then partitioned between the solvent and water to form a water- The lactose and lactose decomposition products contained in the mother's milk are removed by adding it to the mother's milk refined by the Folch partition method and the cold ethanol precipitation, which are separated into the upper methanol layer and the lower chloroform layer. Doing; And
4) separating and purifying breast milk oligosaccharides from breast milk from which lactose and lactose decomposition products of step 3) have been removed. delete delete delete The method of claim 1, wherein the separation and purification of the breast milk oligosaccharide of step 4) is carried out through centrifugation, filtration and lyophilization, the separation of breast milk oligosaccharide from which lactose and lactose decomposition products have been removed without changing the breast milk oligosaccharide. And purification methods. delete The method of claim 1, wherein the lactose decomposition products are glucose, galactose, and ethanol. The method of claim 1,
1) Kluyveromyces lactis with culture removed Mixing 1 part by weight of sodium alginate per 1 part by weight and dropping it in a calcium chloride solution to fix Cluyveromyces lactis;
2) activating the immobilized Kluyveromyces lactis by immersing the immobilized Kluyveromyces lactis of step 1) in the culture medium for 12 hours;
3) The activated immobilized Kluyveromyces lactis of step 2) was collected after only the middle layer after the first centrifugation of breast milk, followed by 2: 1 of chloroform and methanol per 1 part by weight of the middle layer. After adding 4 parts by weight of the mixed solution at a volume ratio of 2 parts by centrifugation, only the supernatant was collected, 3 parts by weight of ethanol was added per 1 part by weight of the supernatant, and then reacted at 4 ° C. for 24 hours, followed by 3rd centrifugation. Separating and collecting the supernatant, and then lyophilizing and adding it to primary purified mother milk dissolved in water to remove lactose and lactose decomposition products contained in mother milk; And
4) separating and purifying the breast milk oligosaccharide by centrifugation, filtration and lyophilization in breast milk from which the lactose and lactose decomposition products of step 3) have been removed; and removing the lactose and lactose decomposition products without changing the milk oligosaccharide. And purification of isolated breast milk oligosaccharides. delete delete

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